The long term goal of this project is to define the molecular basis of agonist affinity and efficacy in the NMDA receptor, a ligand-gated ion channel that belongs to the glutamate receptor family. Activation of NMDA receptors requires binding of two co-agonists, glycine and L-glutamate, to receptor domains in the in the NR1 and NR2 subunits. Occupancy by both agonists initiates a series of molecular events that culminates in opening of the associated ion channel. The objective of this proposal is to identify specific molecular determinants of the interaction of agonists with the NMDA receptor. The recently published crystal structure of the ligand binding domains of a related glutamate receptor (GluR2) predicts which amino acids are in direct contact with the agonists. Preliminary data from our lab suggest the existence of transduction elements in the glycine binding pocket and a highly conserved region in the M3 transmembrane segment. Therefore, the following specific aims are proposed: (1) To identify amino acid residues that determine agonsist affinity and efficacy. Site-directed mutagenesis has identified many amino acid residues whose mutation caused shifts in the agonist dose-response curves. However, such shifts in agonist sensitivity cannot be interpreted unambiguously. A new approach will therefore be used which can distinguish between mutations that affect agonist affinity or efficacy. By using of cysteine-substitution mutagenesis and thiol-specific modifying reagents, the same population of channels can be studied before and after modification. Full and partial agonists will be employed to unequivocally interpret alterations in efficacy and affinity. Parallel experiments using the GluR2 receptor will be used to confirm the structural assignments. (2) To test the hypothesis that the M3 segment is a transduction segment coupling ligand binding to channel opening. The M3 transmembrane segment of glutamate receptors contains a strictly conserved amino acid sequence. Cysteine substitutions in this region identified a residue for which thiol modification results in constitutively active NMDA receptors. Since this modification requires the presence of agonists, it was hypothesized that M3 undergoes a conformational change upon receptor activation and that thiol modification locks the receptor in the active state. These studies will result in a detailed molecular picture of the dynamic change in structure that accompany activation of the NMDA receptor.